USES OF ISOTOPES IN ORGANIC CHEMISTRY' DOROTHY A. SEMENOW and JOHN D. ROBERTS California Institute of Technology, Pasadena. California
INRECENT years, isotopes have become one of the most potent tools available to the organic chemist for determination of reaction mechanisms, quantitative analysis, and elucidation of structures. The purpose of this review is to illustrate a variety of tracer applications by specific examples.
stable isotopes CIaand NISare supplied by the Eastman Kodak Company. The usual standard commercial unit of radioactivity is the millicurie (mc.) which is defined as 3.7 X 10' radioactive disintegrations per second. Most commercially-available GI4-labeled substances have high specific activities (i. e., on the order of one mc. of C14ISOTOPES AND ISOTOPIC ANALYSIS per millimole of compound). This level of activity is Information concerning isotopes of particular im- usrnlly required in work like biochemical feeding exportance to organic chemists is summarized in Table 1. periments where high dilutions of the isotope The common stable isotopes and the most important expect,ed. It is not often economically feasible to synthesize high-activity, specifically-labeled camradioactive isotope(s) are listed. Deuterium (Hz), heavy oxygen (@'), and all of the listed radioactive pounds, since very special vacuum-line and micro isotopes (except C" which has a very short half- techniques are generally necessary. For low specificlife) can be obtained on allocation from the United activity materials (on the order of one mc./mole), States Atomic Energy Commission in a small number the usual organic synthetic techniques may be emof standard chemical forms ( 1 ) . A wide variety of CL4 ployed; syntheses starting from the inexpensive radiot E~~~~~ ~ corn~ and deuterium-labeled organic compounds are avail- active materials supplied by the ~ able from commerical radiochemical laboratories (with mission are often and economicall. AEC a~proval). A few substances containing the Isotopic analyses invariably require rather expensive 1 Contribution NO. 1972 from the Gates Crellin Labors- equipment; in starting a research program considertorim. able care should be exercised to select a suitable TABLE 1 Iaotows of Particula. Irnnortance t o Ormanic Research Atomic number 1
6
Isotope symbol and mass H' H' Ha C" C'2 C1"
Mode of
Abundance, %
Half-life
..
U.02 .
.
12 yezm 20.5 min.
9s:9 1.1 Tracec 99.62 0.38 99.i6 0.20 100
..
6466 years
.. .. .. ..
14'3 days
95:i
8 7 . i davs
75:4
2
24:i 50.5 49.5
x
10; years
.. .. ..
..
..
Be
8'
.. .. Be .. .. .. .. .. Be .. B~
0.015 .9Ub
..
0.10 .. .. .. ..
i.69
o.'k
..
3k' hours
100 ' '
decay
Maximum energ% m. e. ue
8 days
Be
.. .. .. Be .. Be
0.64
.. ..
0.47 0.60
The maximum energy of the emitted particles is important in consideration of possible counting procedures since low-energy particles may not be able to penetrate to the sensitive part of the counter. The maximum penetration ranges in aluminum are about 0.25 cm. for 1.5 m. e. v., and 0.01 cm. for 0.15 m. e. v. pe particles. b Annihilation of the Ba particle by reaction with Be giveas r-ray of 0.5 m. e. v. energy. 0 Natural carbon which is, or was within IO'years, in equilibrium with at.mosphericcarbon dioxide contains a small but detectable amount of C14 ((8). 2
i
~
VOLUME 33, NO. 1, JANUARY 1956
analytical method. Too frequently, economy in analytical instruments is achieved a t the expense of convenient sample preparation and ability to work a t low isotope concentrations. Radioactive isotopes which emit 8-particles (e. g., H3,CL4,and Ilal)are usually analyzed withanionization chamber or Geiger-Miiller counter. With 8-particles of low energy (and penetrating power), highest efficiencies are achieved by counting gaseous samples. For this purpose, ionization-chamber techniques are most effective. It should be remembered that a tenfold increase in counting sensitivity may permit a corresponding reduction in specific activity without loss in counting accuracy. A modern adaptation of scintillation counting is very efficient for &emitters. The radioactive substance to he analyzed is dissolved in a solvent containing an aromatic compound, like terphenyl or anthracene, which produces light upon bombardment with 8particles. The light emission is customarily measured by conversion to electrical pulses with a photomultiplier tube. Thermal noise in the photomultiplier tubes may be reduced by refrigeration and by using two tubes in a coincidence circuit. Analysis of organic compounds for stable heavy is* topes such as Hz, C13, N14, and 0 ' 8 often can be achieved to better than one per cent with a mass spectrograph. I n most cases it is necessary to convert the samples to a standard form such as hydrogen, carbon dioxide, nitrogen, methane, etc., for accurate analyses. Deuterium often can he conveniently determined by infrared spectrophotometric methods since RH and R-D bonds have quite different absorption frequencies. Mixtures of substances which differ only in isotopic hydrogen content customarily follow the Beer-Lambert absorption law very closely. Many of the older deuterium analysis procedures involve precise density or refraction determinations which necessitate extensive and often inconvenient sample purifications. Nuclear magnetic resonance techniques show great promise as a means for analysis of isotopes which have nuclear magnetic moments (e. g., HI, Hz, HS, CL3,and Nl3)(3).
3
neutral "benzyne" intermediate has been considered for this type of reaction (4). OCH.
r
I H'Q'~'" H+
KNH*
YZ
1
OCH.1
I
I
LHY1
KEH*
L
a substituted
"benzyne"
In the corresponding amination of a halobenzene like iodobenzene, the two carbons of the "triple bond" of the intermediate benzyne, CsHa, should be attacked by amide ion with approximately equal probability and rearrangement should occur; such rearrangement would only be detectable through use of an isotopic tracer (4). H NH,
"benzyne"
The logical starting material for an experiment of this type was commercially-available (5) aniline-1-C14 which could be used to make the desired iodobenzene1-C14 and, a t the same time, be employed as material of known isotopic distribution to check possible degradation schemes for locating the position of the C14-label in the amination product. For an unambiguous test of the proposed reaction mechanism, it was necessary to show: (1) that the starting material, aniline-1-C14, ISOTOPES AS TRACERS was labeled as represented; (2) that the iodobenzene Most problems in which isotopes are used as tracers itself did not rearrange in its preparation or in the depend on availability of a starting material with an presence of potassium amide and liquid ammonia; and isotopic label a t a particular position, and determination (3) that the amination product did not rearrange under of where the labeled atom is located in the product. A the reaction conditions. The degradation scheme of equation (3) was emnumber of checks are invariably necessary to avoid ambiguous results. To illustrate the general procedure, ployed to break the original aniline molecules into parts a s~ecificexamde of the use of C14will be discussed in containing specific atoms of the aniline carbon skeleton. The cyclohexanone is seen to contain all six carbon detail. Metallic amides have been long known to react with atoms of the benzene ring with the carhonyl group aromatic halides to yield rearrangement products. corresponding to the 1-position of the starting aniline. For example, o-chloroanisole with sodium amide in The pentamethylenediamine from the hydrazoic acid liquid ammonia yields m-anisidine containing none of reaction contains carbon atoms 2- and 6- of the original the o- or p-isomers. As one working hypothesis, an aniline, while the carbon dioxide represents the carbon elimination-addition mechanism involving a reactive originally a t the 1-position. Thus, the percentage of
JOURNAL OF CHEMICAL EDUCATION NHz
6 0 0 OH
HONO H20,H"
'-. CO!;'
0
OH
II
H*, Pt
-,1 ,
COIH
%02H
. . I ......
jCHn
sCHz
HNs
&
6CH% 1 4 , '
CH? pentamethylenediamine
C02
'CO,
......
......
H2N-LCHn \\
4 ' , '
I
(2 steps)
cyclohexanone
. . I. . .
-
~ C H ~ N H ~ SCH~NH. ' KMnO,
HNI
'CHrNH*
../,
(3)
CHI trimethylenediamine
CHI glutaric acid
the CL4-activityof the cyclohexanone which appears in the pentamethylenediamine is the percentage of rearrangement of CT4from the l-position of the original starting material. The data of Table 2 show that the pentamethylenediamine obtained by degradation of the commercial sample of aniline-l-C14 was inactive. This result indicates that the aniline was labeled only in the l-position as represented and that the degradation scheme was reliable. On the other hand, the aniline from the amination of the labeled iodohenzene contained only 46.4 per cent of aniline-l-C'4, s h o ~ i n gthat the amination product was formed with extensive rearrangement. Further degradation of the pentamethylenediamine gave trimethylenediamine (see equations above) of negligible CL4-activity. The trimethylenedianiine contained the carbons of the 3-, 4-, and 5- positions of the original aniline and the lack of activity in this material indicates that no aniline-3-CI4 was formed in the overall process. This latter result is highly significant since i t eliminates any possibility of substantial rearrangement of the iodobenzene and/or amination product under the reaction conditions. The argument is as follows. If rearrangement of iodobenzene-l-C14and/or aniline-l-C14 occurs t o the respective 2-C14 isomers, then, by the same token, the latter compounds can rearrange to the 3-C14isomers, since all of these isomers do not differ appreciably in chemical properties but only in the position of the isotopic labels. The negligible formation of aniline-3-CL4,as shown by the very low activity of the trimethylenediamine, indicates that the rearrangement actually occurred in the amination reaction. The equivalence of the two "triple-bonded" atoms of the benzyne intermediate suggests that the expected
extent of rearrangement (i. e . , formation of aniline% C14) should be 50.0 per cent if the assumption is made that C12 and C14 atoms react at identical rates in the final ammonia addition step. Actually, as will be noted from the data in Table 2 the observed per cent of rearrangement was 53.0 per cent which is in good, but not perfect, agreement with the above prediction. As will be discussed later, many examples are known in which reactions a t CL4-labeledpositions are as much as 15 per cent slower than a t C'2-labeled positions. About a 12 per cent difference in reaction rates for CL4and CL2atoms (12 per cent isotope effect) would account for the deviation of the observed per cent of rearrangement from that predicted for the symmetrical intermediates. This is well within the range of the common C'4-kinetic isotope effects. If one accepts the conclusion that a symmetrical C6Ha intermediate is involved, then the isotope effect indicates that the intermediate must have a finite lifetime, since the addition of ammonia is slow enough to have discrimination by the ammonia molecules or amide ions between C14and C'2 atoms. The above experiments illustrate a number of important principles for the use of isotopic tracers in determinations of reaction mechanism, many of which are common to almost all tracer problems. A number of other examples of the utility of tracer techniques in organic chemical problems will now be discussed with less complete coverage of details of the synthetic and degradative procedures. STRUCTURE DETERMINATIONS
I n a few cases, isotopic tracers have been employed to eliminate possible strnctures for stable compounds. For example, i t has been shown (6) that a mixed dimer
TABLE 2 C1'-Anslyses of Radioactive Aniline Degradation Pmducts (4) Cyclohezanone, O/o kiie-l-~~' Aniline from reaction of iodobenzene-l-C" with potassium amide a
(100)" (100)'
Penkzmethglenediamine, yo
0.21 1 5 3 . 0 + 0.2'
C O S ,% ~ 96.7 + I d 46.4*0.1
COz from cyclohexanone and HN. GOI from glut.++cid and HNa. Teken as standard. Probably slightly low because of contamination with atmospheric Cop. CO2 from improved isolation procedure with minims1 atmospheric COz oontamination
Trimethylenediamine, %
0.05+0.3
CO.!~. % .-
-
52.4
+ 0.3.
VOLUME 33, NO. 1, JANUARY 1956
of methylketene-l-C'4 and hexylketene does not have a symmetrical cyclohutanedione-1,3 structure as shown in equation (4).
5
ammonia and N1"laheled aniline. This result is not in accord with the symmetrical cyclic structure which would give ammonia containing just half of the NIS.
Ethanolysis of the mixed dimer yields ethyl propionate REACTION MECHANISMS - - - and ethyl caprylate. If the dimer had the symmetrical Bimolecular Nucleophilic Substitution Reactions (SN2). structure, the ethanolysis reaction should give ethyl One of the earliest and most elegant uses of isotopic propionate and ethyl caprylate of equal C14 content since there is an exactly equal probability of splitting tracers for the determination of reaction mechanisms the molecule along the two dotted lines as shown. was the classical demonstration that bimolecular However, the C14-assays showed that 98.5 per cent of nucleophilic displacement reactions proceed with inthe C'4 of the dimer was in the ethyl propionate and version of optical configuration (8). It was shown only 1.5 per cent in the ethyl caprylate. The results that 2-iodooctane underwent exchange with radioof this and further CT4experiments, as well as the active iodide ion in acetone a t the same rate as (+)-2physical properties of the dimeric product, were in iodooctane was converted to the (-)-isomer by oraccord with the conclusion that the dimer was actually dinary iodide ion under the same conditions. Thus, a mixture containing 67 and 33 per cent, respectively, every displacement of ordinary iodine by radioactive of the follow in^ com~oundseach of which can be cleaved iodide ion was accompanied by inversion at the optically active center. by ethanol in only one way. Elimination Reactions. The E2 elimination reacI1 H tions of oreanic halides mav take d a c e hv either a concerted or a stepwise (carbanion) mechantam (equations (9) and (10)). A useful technique for detecting the
-
-
- -7
+ OH
Concerted mechanism: X - b C : H I I
Although infrared absorption and electron diffraction analyses of organic azides support the linear chain formulation for the azide nitrogens, the formerly accepted cyclic structure for phenylazide has only Stepwise mechanism: X - L d : H recently been excluded by isotopic analysis (7). I I HOH
A N's-labeled phenylazide was made by the reaction shown in equation (6).
If the chain structure were correct the labeled nitrogen would be a t the end of the chain and if the symmetrical cyclic structure were correct, then the N'6 atom would have to he geometrically equivalentto one of the ordinary N14 atoms. Treatment of the labeled phenylazide with phenylmagnesium bromide followed by hydrogenolysis of the resulting diazoaminobenzene gave o~dinary
slow
2 +\C=C /+ HOH /
\
+ O~H
fast
shw + g(!de
I =I1
e
L
X
(9)
+\C=C /(10) /
\
presence of the carbanion is to carry out the reaction in a deuterated solvent and determine whether or not exchange to give the deuterated halide is more rapid than elimination. Generally, such exchange is not found with the simple alkyl halides (9).
A
I
I
:
Ha
slow
x-A-L:e
I
xe
I
f\C=d / \
JOURNAL OF CHEMICAL EDUCATION
H
CI
H
CI
-
61
H
@-benzenehexachloride
@-Benzenehexachloride has been found to undergo elimination of hydrogen chloride exceedingly slowly, and this fact has been taken as an indication that eliminations prefer t o occur in a trans-manner because @-benzenehexachloride has all its adjacent hydrogens and ehlorines cis t o one another. It has been proposed that the hexachloride undergoes slow, stepwise ciselimination under the influence of basic reagents by way of a carbanion intermediate. T o determine whether this was the case, the halide was treated with sodium ethoxide in deuterated ethanol (CsHaOD) under conditions where one-half of the material was dehydrochlorinated. The unreacted starting material was isolated and found to contain deuterated 8-benzene hexachloride as expected if a carbanion intermediate were involved in this cis-elimination (10). Favorski Rearrangement. A CL4-tracerstudy (11) has shown that a symmetrical intermediate, sneh as a cyclopropanone, is formed in the Favorski rearrangement of 2-chlorocyclohexanone by ethoxide ion to ethyl cyclopentanecarboxylate. Two reasonable mechanisms may be written for this rearrangement which would lead to products differing in isotopic composition when 2-chlorocyclohexanone-l,2-C~4 is used as a starting material.
compositions predicted for each of these mechanisms and the experimental product composition. The results are clearly in accord with the postulate of a cyclopropanone intermediate for the Favorski rearrangement of a-chlorocyclohexanone. Carbonium Ion Reactions. Much information concerning the nature of carboninm ion type intermediates has been obtained from C14 experiments. Carbonium ions are notorious for their ease of isomerization and tracer studies have been helpful in defining the rearrangement conditions. For example, in the hydrolysis of t-amyl chloride, isotope-position rearrangement could occur via a series of carbonium-ion isomerizations as shown in equation (15). Such isomerisations do not
occur under conditions as mild as those in aqueous hydrolysis, but isotope-position rearrangements have been observed on treatment of labeled t-amyl chlorides with aluminum chloride (18). Very extensive re-
Table 3 contains a comparison between the product Expected and Observed Isotopie I)istFibutions of Ethyl Cyclopentanecarboxylate from Favorski Rearrangement of 2-Chlorocyolohexanone-1.2-C:' (11) Ezpected Ezpected for
f o ~
Found -
50 25
25
VOLUME 33. NO. 1. JANUARY 1956
arrangement was found in the conversion of C1*labeled isopentane to t-amyl bromide (13) by the hydrogen-halogen exchange reaction of Bartlett, Condon, and Schneider (14).
classical cmbocation; nonclassical carbocation; positive charge rela- positive charge dispersed tively localized over several atoms
It has been well established that the 3-phenyl-2butyl cation is substantially less stable than the nonclassical phenonium ion and in many reactions the phenonium ion appears to be formed directly in the ionization process (16). In an investigation of the effect of structure on the relative stabilities of classical
$2 X
Considerable research has recently been done to determine which varieties of carbonium ions exist most favorably in nonclassical bridged structures as shown in equation (18). A large number of experimental techniques have been applied to this type of problem and it will suffice to give two examples of isotopic tracer experiments.
a
=
Cltlaheled phenyl group
=
phenonium ion
p-toluenesulfonate, halide, etc.
and nonclassical cations, it was found that only classical carbonium-ion intermediates can account for rearrangements accompanying the acetolysis of 1,2,2-triphenylethyl acetate. The specific reaction rate constants for reactions (20), (21), and (22) were measured and found to be equal to within experimental error (16). It can only be concluded from the equality of the reaction rates that every time a labeled l,2,2-triphenylethyl cation is generated in this system it becomes completely equilibrated with all of its isomeric forms before reverting to ester. At equilibrium, the ratios
JOURNAL OF CHEMICAL EDUCATION
/
w-CHI
0-CCH,
a
of the concentrations of ions A and B above must he 2.00 because of the statistical factor, i. e., there being two phenyl groups on one side of the molecule and only one on the other. If a nonclassical phenonium ion andogous to that formed in the reactions of 3-phenyl2-butyl derivatives were the stable reactions intermediate (equation (24)), the rate of scrambling of the phenyl groups would be three-fourths + 2 / g = 3/1) as fast as predicted by equation (23). Investigation of the reaction of Cr4-labeled cyclo-
0
slow
+
L C -11C H ,
0
propylcarbinylamine with nitrous acid reveals that cyclobutanol is formed by a process in which thethree methylene groups of the starting material achieve a very high degree of equivalence (17). This result is well (though not uniquely) accounted for by a pyramidal nonclassical cationic intermediate as shown in equation (25). Claisen Rearrangement. Isotopic tracer studies (18) of the o- and p- Claisen rearrangements strongly indicate that these reactions proceed by cyclic mechanisms
VOLUME 33, NO. I , JANLIARY 1956
9
rather than by stepwise processes involving symmetrical carbonium-ion intermediates. The results expected for each mechanism in the rearrangement of allyl-3-C1" p-tolyl ether are shown in equations (20) and (27). It was found experimentally that all of the activity in the ally1 group of the o-allyl-p-cresol was contained in the carbon attached to the aromatic ring (18). Benzilic Acid Rearrangement. Tracer experiments have aided in the elucidation of the mechanism of the benailic acid rearrangement. Oxygen exchange of benzil with sodium hydroxide- 018under the conditions employed in the rearrangements demonstrated a rapid pre-equilibrium of the following type.
which aryl group migrates in the rearrangement of an unsymmetrical benail (21). Degradation of the rearrangement product of l-(p-methoxyphenyl)-2-phenyl-1,2-ethanedione-l-C1" showed that the p-methoxyphenyl group migrates about half as fast as the phenyl group. This result is particularly interesting since, in other rearrangements, pmethoxyphenyl groups migrate to electron-deficient centers far more readily than do phenyl groups. The dominant factor in determining which group migrates in a benzilic acid rearrangement appears to he the relative stabilities of the intermediate ions 'shown in equation (31). Since intermediate A is expected to Fe
The benzil was heated very briefly with sodium hydroxide in a water-O's-methanol medium. The reaction time was sufficiently short so that rearrangement did not occur to an appreciable extent and the decrease in 0ls content of the recovered water from the solvent indicated that the above equilibria were completely established (19). The benailic acid rearrangement is first-order in benzil and first-order in hydroxide ion (20); the following mechanism (29) which can be written for the rearrangement is consistent with both the kinetics and 018-exchange experiments. Radioactive carbon has been used to determine
of lower energy than B owing to the smaller electrondonating tendency of phenyl compared to pmethoxyphenyl, A should be present in higher concentration than B. Hence, phenyl migration can occur more easily than p-methoxyphenyl migration, provided that the transition states for the slow rearranging steps are similar in structure to A or B, respectively. REACTIVITY STUDIES
Exchange Rates. Numerous experiments have utilized hydrogen-deuterium exchange in acid or basic media to probe for sites of high and low electron density.
OH phenyl migration
0
0
C H 3 0 a - ! Y - l ! - a -
-\
p-methoxyphenyl migration
OH
OH
p-methoxyphenyl migration
JOURNAL OF CHEMICAL EDUCATION
*
38" NHa
-NH2
d D
ae
$
rnKH%
e
H
D
Thus, aniline which is expected to have relatively high electron density a t the o- and p- positions exchanges its o- and p- hydrogens rapidly in acidic deuterium oxide solutions (22). This technique has provided a vivid argument for the proposition that second- and higher-row elements can have important contributions of resonance forms involving expansion of their valence shells beyond eight The argument is based On the kding that trimethylsulfonium but not tetramethylammonium ions rapidly exchange methyl hydrogens for deuterium CH, cH,-i-cHa
-O"D
excess DIO
D
The relative reaction rates for the various suhstituents are as follows: o-F >> o-CF3 > rn-CF8 > 20,000 3000 70
-
o-OCH8 60
-
p-CF8 > m-F > 50 25
p-F > m-OCH3 > p-OCH* (1.0) 0.01 -0.001
~h~ experimental results appear to he best interpreted in terms of inductive and field effects which fall off very rapidly with increasing distance the stituent and the reaction (24).
CDa
cD8-bcD3
(33)
KINETIC ISOTOPE EFFECTS
Isotopic tracer techniques are commonly based on the premise that a tracer atom is the exact chemical CH. aI O"D equivalent of the normal atom it replaces. Actually, CH,-A--CH, no exchange I excess DIO (34) isotopic atoms often display differences in chemical CH1 reactivity which are termed kinetic isotope effects. of the Thus, the difference in the rates a t which C-H and in basic solution (23). ~h~ extra bonds are may he a deuterium conjugate base of the trimethylsulfoninm ion is easily envisioned in terms of involving the posi. !sotope effect. The magnitude of such effects depends way On the percentage difference tive sulfur atom as shown in the resonance forms of !" a ~n masses of the isotopes involved. equation (35). Theoretical Basis. We consider first possible effects CHa of isotopic substitution on bond energies. If the as1 -He sumption is made that only differences of nuclear C H A -.C H , . @ masses are involved then we can consider the electrical binding energies between isotopic atoms such as C CHs I (36) -H and C-D bonds to be equal. In this situation, CHJ-5-CH2c----t CHsS=CH2 .. the potential energy curves for breaking C-H and C-D bonds will be the same (see the figure). ThereThe rates of exchange of benzenoid deuteriums lo- fore, any difference in the energies required for bondcakd ortho, mela, and Para to fluorine, trifluoromethyl, breaking must be associated with a difference in vihraand methoxyl groups have been determined in solutions tional energies of the starting materials. since at of ~otassiumamide in liquid ammonia (24). Exchange moderate temperatures almost all of the bonds will be is exceptionally fast with o-deukrofluorobenzene, in their lowest vibrational states, they will have the the reaction being complete in less than 15 seconds. same vibrational energy as a t absolute zero (i.e., the D H zero-point vibrational energy). To a reasonable degree I of approximation, the zero point stretching vibrational (36) energy Eo (which alone will be considered in the sequel) for a bond between two atoms is given by the following X x X ~ equation:
--
CFD
-
yHa
]
VOLUME 33, NO. 1, JANWARY 1956
11
malonic acid in the ratedetermining step. The isotope effectfor malonic acid-l-CI4 is approximately twice that found for C13 (27). where h = Planck's constant (erg sec.); k = bond A large number of CIGsotope effects have been stretching force constant (dyne cm.?); u = reduced measured and correlated with reaction mechanisms mass = mLmz where ml and mz are masses of the (28). A notably elegant study has been made of the m~ mz' change in C1%otope effect in the alkaline saponificaindividual atoms. Now all of the factors in the tion of carboxyl-labeled ethyl benzoates with various above equation for C-H and C-D bonds will be substituents in the m t a - and p a r 5 positions (29). :I Deuterium Isotope 3ffects. As would be expected the same except for .$ which will have the values from the theoretical discussion given above, more proI,. nounced isotope effects are shown by deuterium than 2+12 d2 respectively. -1 and -, by CL3or C14. Thus, the gross rate of chromic acid 1 . 12 2 oxidation of isopropyl alcohol is about seven times that of Since EO is directly proportional to the zero- or-deuteroisopropyl alcohol. I n contrast, there is less .. than 10 per cent differencebetween the rates of chromic point vibrational energy for C-H bonds is greater acid oxidation of isopropyl alcohol and phexadeuterothan for C-D bonds. Correction of the potential isopropyl alcohol. These results establish beyond energy curves (see the figure) for the zero-point energy question that the bond to the a-hydrogen is broken in shows that net energy for breaking a C-H bond the slow step of the chromic acid oxidation of secondary should be less than that for a C-D bond because the dcohols to ketones (SO). C-H compound has more vibrational energy, i. e., is An interesting secondary deuterium isotope effect less stable. On this basis, a reaction in which a C-H has been reported for the free-radical polymerization of bond is broken might be expected to proceed more allyl acetate (31). Polymerization of ordinary allyl rapidly than the corresponding reaction with a C--D acetate gives a relatively short-chain polymer because bond. However, it must be noted that the foregoing addition of monomer molecules to the growing chain has zero-point energy effect may only be rigorously applied to compete with hydrogen atom abstraction which terto energy differences between starting materials and minates one chain and starts a new one. This chainfinal products. The situation is more complex for transfer reaction is markedly slowed by having deutepredictions of reaction rates since, besides the zero- rium in place of hydrogen on the a-carbon of the allyl point energy difference between reactants and transition acetate. Thus, in the polymerization of orp-dideuterostate complex, there is an isotope effect on the rate allyl acetate, a given chain adds on more monomer units constant for decay of the activated state (26). before chain-transfer occurs, giving a higher molecular Differences in reduced mass will be small when the weight polymer. percentage differences in the masses of two isotopes are small. Thus, with C12H and C14-H bonds, there is only about a one per cent difference in the reduced mass. In such cases, the kinetic isotopic effect will be much less prominent than VIBRATIONAL ENERGY LEVELS with hydrogen and deuterium. Kinetic isotope effects can be extremely helpful in determinations of reaction mechanisms because wherever such an effeet is found, of the order of magnitude predicted from zero-point energies dii- C3 ferences, there is a strong supposition that a bond involving the isotope is made or W broken in the slow step of the reaction. Thus, isotope effects may he used to dis- W tinguish between the fast and slow steps of a complex reaction mechanism. Carbon Isotope Effects. The ratio of thermal decarb~x~lation rates or ordinary I I malonic acid and malonic acid-l-CIS has I been s h ~ w n(26) to correspond to a ratio of rates of breaking of C12-C12 and C1-CIS bonds of 1.037. This effect may rc-~ 'c-D be regard* as evidence for the Bchametic Potential-enDiagrmrm b C--H urd C--D Bond* T&en u Diatomis of the bond between C-1 and C-2 of the MOL=U~-
+
d E
&-
-
.\li,
t
>
z
JOURNAL OF CHEMICAL EDUCATION addition R--CH-CH. kH20Ac growing chain radical
-+
i
I
11.
0
CIIp==CH=CH
bA0
CHFCH
__
~ H ~ O A ~
CHFCH-CH-CHI-CH.
-.I
UAC
R--CH2-CH-CH2-CH .
/-CHSOAC abstraction \R--CHrCH2 (chain trsnsler)
CH,=CH
L. -.
UdnxUAC
(38)
new growing chain radical
Deuterium studies (4) provide strong support for the elimination-addition mechanism in the rearranging aminatious of halobenzenes with the metallic amides which were discussed earlier. The formation of benzyne from a halobenzene may occur by either of the following mechanisms.
-
Lac -
hHmc
(37)
0
i CHz=CH=CH (&HgOAe O I Ac
dinary ammonia, exchange was found to occur a t a rate comparable to elimination suggesting that this halobenzene reacts by the stepwise mechanism(41). P-Deutero-t-amyl chlorides, containing two, six, and eieht deuterium atoms ~ e molecule. r exhibit subs k i a l isotope effects upin solvolysis 'in 80 per cent aqueous ethanol (59). he octadentero compound solvolyzes approximately two-fifths as fast as the undeuterated chloride. The less extensively deutewted compounds have intermediate reactivities approximately in ~roportionto the number of atoms of deuCH~-&-CH~CH~
CH~LCD~C A1 0.71
A1 Relative k, 1.00 CDI
~ D x
I
terium on the @-carbons. These isotope effects have been interpreted in terms of solvation of the incipient A kinetic isotope effect of approximately seven is pre- carbonium-ion intermediates a t the p-hydrogen (or dicted for the formation of benzyne by mechanism deuterium) atoms and in terms of purely hypercon(40) when o-deuterohalohenzenes are used as the start- jugative stabilization of the intermediate (St?). Both ing material. Mechanism (39) may or may not lead to explanations postulate stretching of the p-C-H (or an isotope effect depending on the ratio of the rate for C-D) bonds in going from the starting materials to loss of a halide ion from the carbanion to the rate of re- the solvolysis transitions states. version to the starting material by abstraction of a proton from the solvent. The smaller this ratio, the MOLECULAR ASYMMETRY DUE TO ISOTOPES smaller will be the observed differences in the rates of For many years attempts were made to prepare reaction of the aromatic C H and C-D bonds. The compounds with detectable optical rotations where the kinetic isotope effects for these reactions have been only asymmetry is due to replacement of hydrogen by determined by comparing the relative reaction rates of deuterium. The first successful preparation of a o-deuterohalobenzenes and ordinary halobenzenes with demonstrably asymmetric substance of this type was lithium diethylamide in ether and with potassium amide that of 2,3-dideutero-trans-menthaneby hydrogenation in liquid ammonia. With o-deuterobromobenzene and of trans-2-menthene with deuterium (55). either lithium diethylamide in ether or potassium amide in liquid ammonia, the observed isotope effects were 5.6 + 0.1. showing - that the o-hydrogen is removed in the rate-&nnilling step of t h r aminntion renetions (mrdianism(lOj,. With o-(le~ltcrorhl~~n,I,rr~zerlr i n ortrans are -0.14 + 0.01'
mm
trans +31.09"
trans ao 0.00
O.O1°
The criteria for the reliability of this experiment were (1) elaborate purification, (2) reproducibility of the optical activity, and (3) controls in which the trans-2menthene was reduced with hydrogen to give a completely inactive trans-menthane.
VOLUME 33. NO. 1. JANUARY 1956
13
A fourth criterion was recently employed to demonstrate the presence of optical activity in (-)-1-bromohutane-1-D ($4). Measurements were made of the rate of racemization of the optically active bromide in the presence of lithium bromide in 90 per cent acetoue10 per cent water. The rate of racemization was found to be the same as the rate of exchange of ordinary n-bntyl bromide with radioactive bromide ion (as a lithium salt) in the same solvent. The equivalence of Br
D
+ J%*
n-C4H,-Br
kr w n-C4HQ-Br*
+ Bre
when used to reduce ordinary acetaldehyde. This monodeuterated ethanol reacted with diphosphopyriH
0 CH,~-H
D /CONH1
M I
R L
ADH
(44)
k, = kn within experimental error
these reaction rates is strong evidence for asymmetry of the deutero halide a t the primary carbon atom. Furthermore, these experiments indicate that bimolecnlar nncleophilic displacements on primary halides proceed with inversion of configuration. Perhaps the most unequivocal evidence for the existence of enantiomers of compounds of the type RIRsCHD has been obtained through the stereospecific reaction catalyzed by the enzyme, yeast alcohol dehydrogenase (ADH) (35). In the presence of ADH, diphosphopyridine nucleotide oxidizes ethanol t.o acetaldehyde and is converted to reduced diphospJ3ONH'
+ CHaCHIOH
ADH
I
dine uncleotide to give acetaldehyde containing no deuterium. These reactions show that the ethanol1-D was asymmetric because only deuterium was transferred from the a-carbon of the ethanol to the nucleotide in the oxidation reaction. The other enantiomer of ethanol-1-D was prepared by reduction of acetaldehyde-1-D with the reduced nucleotide containing no deuterium. This alcohol was oxidized by the nucleotide to produce acetaldehyde-1-D containing all of the deuterium. Decisive evidence that the two 1-deuteroethanols were enantiomers was obtained by transforming one into the other through an S 2, inverting hydrolysis of p-toluenesulfonyl ester with sodium hydroxide solution.
R diphosphopyridine nucleotide H
H
M""""
+ CH,CHO + He
(45)
I
R reduced diphosphopyridine nucleotide
phopyridine nucleotide. Reduced monodenterodiphosphopyridine nncleotide was synthesized from 1,ldideuteroethanol by enzymatic reaction (46). D
ADH
1
m,
D
The deuterated reduced nucleotide yielded ethanol-1-D
ISOTOPE DILUTION ANALYSIS
Quantitative analysis of organic components in a reaction mixture is frequently difficult if the compound to be analyzed is present in small quantity and/or is similar to the other components of the mixture. With isotope dilution procedures, quantitative isolation of a compound is not necessary for analysis ($6). The only requirement is a sufficiently pure sample for isotopic assay. Isotope dilution analysis to obtain the number of moles x of an organic compound in a mixture may be accomplished by adding a given number of moles m of the same compound labeled with an isotope of know specific activity So. After thorough mixing, a sample of the compound is isolated from the mixture, purified, and analyzed for radioactivity. The new lower specific activity is S. The number of moles of
JOURNAL OF CHEMICAL EDUCATION TOPLEY, AND J. WEISS,J. Chem. Soe., 1935, p. 1525. (9) SKELL, P. S.. AND C. R. HAUSER, 3.Am. C h a . Soc.. 67,1661 (1945); D. G . HILL. W. A. JUDGE,P. S. SKELL,8. W. KANMR,AND C. R. HAUSER,ibid., 74, 5599 (1952); D. G. HILL.B. STEWART, S. W. KANTOR, W. A. JUDGE. A related method may be used for analysis of the AND C. R. HAUSER, ibid., 76, 5129 (1954). components of reaction mixtures where the starting (10) CRISTOL,S. J., ibid., 69, 338 (1947); S. J. CRISTOL,N. L. HAUSE,AND J. S. MEEK. ihid., 73, 674 (1951); S. J. material may be labeled with a suitable isotope. An CRISTOL, AND D. D. FIX, ibid., 75,2647 (1953). example is the measurement of the distribution of (11) LOFTFIELD, R. B., ibid., 73,4707 (1951). isomers in nitration of chloro-, bromo-, and iodobenzenes (12) ROBERTS,J. D., R. E. McMAnoN, AND J. S. HINE,ibid., 72, containing radioactive halogens (5'7). The analyses 4237 (1950). ROBERA, J. D., AND G. R. COIMOR, ibid., 74,3586 (1952). were carried out through the following steps: first, adBARTLETT, P. D., F. E. CONDON, AND A. SCHNEIDER, ibid., dition of known quantities of the inactive forms of each 66, 1531 (1944). mononitrohalobenzene isomer to fractional parts of the CRAM,D. J., ibid., 71, 3863, 3875 (1950); 74. 2129 (1952). reaction mixture; second, separation, and purification BONNER, W. A,, AND C. J. COLLINS, ibid., 77, 99 (1955). of the diluted isomers to radiochemical purity; and ROBERTS, J. D., AND R. H. MAZUR, ibid., 73,3542 (1951). SCHMID. H.. AND K. SCHMID.Heh. Chim. Acta. 35. 1879 third, comparisons of the radioactivities of the diluted ( m i ) ; 36,687 (1953). forms with the activities of the starting materials. In ROBERTS,I., A N D H. C. UREY,3. Am. Chem. Soc., 60, 880 this manner, even the small (0.5-2.0 per cent) fractions (19381. ~ ~ ~ - , . of meta- isomers were determined with reasonable (20) WESTHEIMER, F. H., ibid., 58,2209 (1936). J. D., D. R. SMITH,A N D C. C. LEE, ibid,, 73, 618 (21) ROBERTS, accuracy. 11051> ,--"-,. A related technique has been used to analyze mixtures (22) INGOLD, C. K., C. G. RAISIN,AND C. L. WILSON, 3. Chem. of amino acids in protein hydrolyzates (58). The mixed Sac., 1936, p. 915; A. P. BEST,A N D C. L. WILSON,ibid., amino acids are converted to derivatives of p-iodo1938, p. 28; C. R. BAILEY,J . B. HALE,N. HERZFELD, C. K. INGOLD, A. H. HECKIE,AND H. G. POOLE,ihid., benzenesulfonyl chloride containing radioactive iodine. 1946, p. 255. The resulting mixture of p-iodobenzenesulfonamide W. V. E., A N D A. K. HOFFMANN, ibid., 77, 4540 derivatives of the amino acids are diluted with known (23) DOERING, (195.51~ ~-~-.,. amounts of the corresponding labeled derivatives and (24) HALL,G . E., R. PICCOLINI, A N D J. D. ROBERTS, 3. Am. separated and purified for radiochemical analysis. Chem. Soc., In press. (25) BIGELEISEN, J., 3. Chem. Phys., 17, 675 (1949). CHROMATOGRAPHY J., AND L. FRIEDMAN, ibid., 17, 998 (1949); (26) BIGELEISEN, P. E. YANKNICH. AND A. L. PROMISLOW. J. Am. Chem. Mixtures of colorless compounds have been sep* Soc., 76, 4648 (1954). rated by chromatography after conversion to deriva- (27) ROPP,G. A., AND V. F. RAAEN,ibid., 74, 4992 (1952). tives labeled with radioactive atoms. Each component (28) ROPP,G. A,, Nmleonics, 10,22 (1952); G. A. Pam, V. F. RAAEN,AND A. J. WEINBERGER, 3. Am. Chem. Soc., 75, on the column can be located and quantitatively es3694 (1953). timated with a scintillation counter. This method has G. A., AND V. F. RAAEN, 3. Chem. Phys., 2 2 , 1223 been employed recently in the separation of steroids as (29) ROPP, 114'rA) *,. their piodo13'-benzoates (39). (30) WESTHEIMER, F. H., AND N. NICOLAIDES, 3. Am. C h m . Much interesting and helpful practical information SOC.,71, 25 (1949); A. LEO AND F. H. WESTHEIMER, ibid., 74, 4383 (1952); M. COHENAND F. H. WESTon the use of tracers in organic chemistry and biology ibid., 74, 4387 (1952). HEIMER, has been compiled by Calvin, Heidelberger, Reid, (31) BARTLETT, P. D., A N D F. A. TATE,ibid., 75, 91 (1953). Tolbert, and Yankwich (do), and Kamen (41). (32) SHINER,V. J., JR., ibid., 75, 2925 (1953); E. S. LEWIS, AND C. E. BOOZER, ibid., 76, 791 (1954). LITERATURE CITED E. R.. AND A. G. PINKUS, ibid., 71,1786 (1949). (33) ALEXANDER. (1) "Isotopes," Catalog and price List, Oak Ridge National (34) STREITWEISER. A., ibid., 75,5014 (1953); see also Abstracts Laborstory, Oak Ridge, Tennessee. of New Ydrk American Chemicsl Society iMeeting, (2) ANDERSON, E. C., W. F. LIBBY.S. WEINHOUBE, A. F. REID, September 12-17, 1954, p. 2, sect. 0. A. D. KIRSHENBAUM, AND A. V. GROSSE,Science, 105, A N D B. VENNESLAND, (35) LOENUS,F. A,, F. H. WESTHEIMER, 576 (1947). ibid.. 75. 5018 (19531. , J. N., Anal. Chem., 26, 1400 (1954). (3) SHOOLERY, (36) HENRIPWE;,F. C., AND F. MARGNETTI, Ind. Eng. Chem., (4) ROBERTS. J. D.. H. E. SIMMONS. JR.. L. A. CARLSMITH. AND Anal. ed., 18, 476 (1946). C. W.'VAU~HAN, 3. Am. Chem.'~oc.,75, 3290 (1953); (37) ROBERTS.J. D., J. K. SANFORD. F. L. J. SIXU, H. J. D. ROBERTS,D. A. SEMENON, H. E. SIMMONS. JR., CERFONTNN, AND R. ZAGT,3. Am. Chem. Soc., 76, 4525 AND L. A. CARLSMITH, ibid., in press. (1954). FIELDS,M., M. A. LEAFPER,AND J. ROHAN,Science, 109, (38) KESTON,A. S., 8. UDENFRIEND, AND R. K. CANNAN, ibid., 35 (1949); M. FIELDS,J. GIBBS,AND D. E. WALZ,ibid., 71, 249 (1949). 112, 59 (1950). (39) STOKES,W. M., F. C. HICKEY,AND W. A. FISH,ibid., 76, ROBERTS, J . D., R. AWSTRONG, R. F. TRIMBLE, JR., A N D 5174 (1954). M. BURG,J . Am. C h m . Soc.. 71, 843 (1949). J. C. REID,B. M. TOLBERT, (40) CALVIN;M.. C. HEIDELRERGER, CLWSIUS,K., AND H. R. WEISSER,Heh. Chim. Ada, 35,1548 AND P. E. YANKWICH, '?mtopic Carbon," John Wiley & ibid., 37, 383 (1952); K. CLUSIUS,AND H. H~~RZELER, Sons, Inc., New York, 1949. 119541 (41) KAXEN,M., "Radioaotive Tracers in Biology," Academic ,----,(8) HUGHES, E. D., F. J U L ~ S B ~ G ES.R MASTERMAN, , B. Press, Inc., New York, 1941.
the compound originally present in the mixture may then be calculated from the expression:
. .
.
~~
